Recently, the cDNA of TR from human placenta was cloned and sequenced. An additional redox pair at the C-terminus of the enzyme was found to consist of Cys-Secys. The C-terminus extension and the C-terminus redox pair account for the wide substrate specificity and are the main features that differentiate mammalian thioredoxin reductase from the smaller prokaryotic and yeast enzymes. To further study the role of selenocysteine in electron transfer from NADPH to substrate, we overexpressed the Secys/Cys mutant of human cytosolic TrxR1 in E. coli (TGA codon changed to TGT). To overexpress human TrxR1 containing selenocysteine, SECIS required for selenocysteine incorporation in E. coli formate dehydrogenase (fdh) or minimal SECIS (Z. Liu et al. 1998) was inserted downstream of the TGA codon after the gene-specific sequence. Wild type without SECIS, wild type with SECIS or minimal SECIS, and the Sec/Cys TrxR1 mutant were amplified by PCR and ligated to different plasmids to enhance solubilization and the purification yield of expressed proteins. E. coli cells were transformed with the plasmids containing these genes with and without the SECIS in the 3 untranslated region. Most of the expressed protein appeared in inclusion bodies. In order to increase solubilization and correct folding of the expressed TR, induction was done with different IPTG concentrations at decreased temperatures, transformed cells were grown in different media (LB and very rich LB) containing molecular chaperones such as glycerol or betaine/sorbitol under osmotic stress and. E. coli cells were cotransformed with TR and the E. coli chaperones GroES and GroEL. The solubility of TR and the extent of FAD incorporation increased when cells were grown at low temperature (14-25 C) in the very rich LB media with low concentrations of IPTG used for induction. These optimized conditions allowed the purification of milligram quantities of recombinant protein per liter of growth medium. Most of the soluble protein formed dimers under reducing conditions. Specific activity of the Secys/Cys mutant is about 1 percent of that for the native rat liver enzyme (as measured by DTNB reduction). Wild type protein expressed in E. coli was low even with the stem-loop containing plasmids. Preliminary mass spectrometry results suggest that the UGA codon was mostly translated as a stop codon. Selenium incorporation depended on the presence of the SECIS or the minimal SECIS element. Based on 75-Se content only about 2 percent of purified fusion protein contained the selenium moiety, most likely as selenocysteine. - selenocysteine, SECIS, recombinant